A color-producing Streptomyces XS-4 and its application

Abstract

This invention discloses a polychromogenic Streptomyces XS-4 and its applications, relating to the field of screening antagonistic bacteria for anthracnose in tea trees. The polychromogenic Streptomyces XS-4 was deposited on October 19, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231945, and classified as Streptomyces polychromo genes XS-4. This invention uses *C. camelliae*, the causal agent of tea anthracnose, as an indicator fungus. A *Streptomyces* strain XS-4 with good antagonistic effects was isolated and screened from tea garden habitats using the plate confrontation method. Species identification was performed through morphological, physiological, and biochemical characteristics and 16S rRNA gene sequence analysis. The antibacterial properties of the antagonistic bacterium XS-4 against *C. camelliae* were analyzed through antibacterial spectrum determination, scanning electron microscopy, screening using sterile fermentation broth and culture time, determination of the stability of antibacterial activity in sterile fermentation broth, production of extracellular degradation enzymes, and detection of antibacterial substance synthesis genes. This provides biocontrol resources and technical support for the prevention and control of tea anthracnose.

Description

Technical Field

[0001] This invention relates to the field of screening technology for antagonistic bacteria against anthracnose in tea trees, and more specifically to a color-producing Streptomyces XS-4 and its applications. Background Technology

[0002] *Colletotrichum camelliae*, the causal agent of tea anthracnose, is one of the main pathogens of tea anthracnose. It affects most tea varieties in my country and occurs in most tea-producing areas, making it the primary pathogen causing tea anthracnose in the country. Tea anthracnose not only reduces tea yield but also severely impacts tea quality. Currently, chemical agents are mainly used for the control of tea anthracnose. However, the use of chemical agents poses certain risks to both the tea plants and the environment, and can lead to drug resistance in tea plants and kill beneficial organisms. Utilizing beneficial microorganisms to control plant diseases has become an important research direction in plant disease management. While there are some research reports on the biological control of tea anthracnose using beneficial microorganisms, research reports on the use of actinomycetes for the control of tea anthracnose are relatively few.

[0003] Actinomycetes are a type of microbial resource with important economic and biological research value. Among actinomycetes, Streptomyces are the most widely used, and many research reports have shown the application value of Streptomyces in plant diseases. For example, *Streptomyces samsunensi* has a strong antagonistic effect on rubber brown root rot; the fermentation broth of *Streptomyces toxytricini* significantly inhibits the mycelial growth and spore germination of cucumber wilt fungus; *Streptomyces 3-22* has a strong antagonistic effect on cherry leaf spot, with its sterile filtrate showing an inhibition rate of over 80%; *Streptomyces* have the ability to produce a variety of bioactive substances. Studies have shown that *Streptomyces griseorubiginosus* can produce a variety of antibacterial substances, and *Streptomyces violaceorubidus* can not only produce extracellular antibacterial substances but also has the ability to produce a variety of degrading enzymes; the antibacterial active substances in the fermentation broth of *S. lunalinharesii* have good thermal stability, and after treatment below 120℃, the inhibition rate against *Colletotrichum horii* is over 95%.

[0004] Therefore, how to screen for a biocontrol strain of tea anthrax is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] One of the objectives of this invention is to provide a Streptomyces polychromogenes XS-4, which was deposited on October 19, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231945 and classified as Streptomyces polychromogenesXS-4.

[0006] Another objective of this invention is to provide the application of the aseptic fermentation broth, bacterial suspension, and bacterial agent of the aforementioned Streptomyces multiflora XS-4 in the prevention and control of anthracnose in tea trees.

[0007] In the above applications, Streptomyces multiflora XS-4 possesses a polyketide synthase-related gene, thereby producing related antimicrobial substances.

[0008] The present invention also claims protection for the use of Streptomyces xS-4 in the control of other plant pathogens, including Phytophthora blight of strawberry, Anthracnose of Polygonatum sibiricum, Scab of citrus, Brown spot of eggplant, Fusarium wilt of pepper, Fusarium wilt of water chestnut, Fusarium wilt of cotton, and large leaf spot of corn.

[0009] In the above applications, Streptomyces xS-4 can produce amylase, protease, β-1,3-glucanase and cellulase.

[0010] As can be seen from the above technical solution, compared with the prior art, this invention uses *C. camelliae*, the causal agent of tea anthracnose, as an indicator fungus. A *Streptomyces* strain XS-4 with good antagonistic effect was isolated and screened from the tea garden habitat using the plate confrontation method. Species identification was performed through morphological, physiological, and biochemical characteristics and 16S rRNA gene sequence analysis. The antibacterial characteristics of the antagonistic bacterium XS-4 against *C. camelliae* were analyzed through antibacterial spectrum determination, scanning electron microscopy, screening using sterile fermentation broth and culture time, determination of the stability of antibacterial activity in sterile fermentation broth, production of extracellular degradation enzymes, and detection of antibacterial substance synthesis genes. This aims to provide a certain scientific theoretical basis for the future biological control of tea anthracnose. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0012] Figure 1The attached figure shows the inhibitory effect of strain XS-4 on tea anthracnose bacteria on PDA. A: Treatment group; B: Control group (CK).

[0013] Figure 2 The attached figure shows the morphological characteristics of strain XS-4 on Gao's No. 1 medium;

[0014] Figure 3 The attached figure shows a phylogenetic tree of strain XS-4 constructed based on the 16S RNA gene sequence;

[0015] Figure 4 The attached figure shows the inhibitory effect of strain XS-4 on the mycelial growth of tea anthracnose fungus; A and B are control groups (CK), and C and D are treatment groups.

[0016] Figure 5 The attached figures show the antibacterial activity of sterile fermentation broth of strain XS-4 on *Bacillus anthracis* in different fermentation media (top) and the antibacterial activity of sterile fermentation broth of strain XS-4 on *Bacillus anthracis* in different fermentation media (bottom); where A. control; B. corn medium; C. YEPD medium; D. millet medium; E. ISP2 medium; F. YG medium; G. LB medium; H. PDB medium; I. KMB medium; J. Gao's No. 1 medium;

[0017] Figure 6 The attached figure shows the antibacterial activity of sterile fermentation broth of strain XS-4 at different fermentation times against tea anthracnose bacterium.

[0018] Figure 7 The attached figure shows the stability determination of the antibacterial activity of the aseptic fermentation filtrate of strain XS-4 against *Bacillus anthracis*. A shows the effect of pH on antibacterial activity; B shows the effect of UV irradiation time on antibacterial activity; C shows the effect of temperature on antibacterial activity; and D shows the effect of proteinase K and pepsin on antibacterial activity.

[0019] Figure 8 The attached figure shows the assay of fungal cell wall degrading enzymes produced by strain XS-4; A. amylase; B. protease; C. β-1,3-glucanase; D. cellulase;

[0020] Figure 9 The attached figure shows the PCR amplification results of the nrp, pks-Ⅰ, and pks-Ⅱ genes of strain XS-4. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: Isolation and Screening of Antagonistic Strains

[0023] Streptomyces was isolated from the tested soil samples using the dilution plating method. 1 g of soil sample was added to a test tube containing 9 mL of sterile water, shaken on a high-speed shaker for several minutes, and then serially diluted with sterile water to a concentration of 10. -6 , absorb 10 -3 ~10 -6 100 μL of each grade of bacterial suspension was spread onto Gao's No. 1 medium plates, with each treatment repeated three times. After incubation at 28°C upside down for 7 days, single colonies with different morphologies were picked, purified, and preserved. The plate confrontation method was used to screen for Streptomyces strains antagonistic to *C. camelliae*. Test strains were inoculated on both sides of a PDA plate 25 mm from the center. After incubation at 28°C for 2 days, a 5 mm *C. camelliae* mycelial cake was inoculated in the center of the plate. After another 5 days of incubation, the diameter of the inhibition zone was measured, and the inhibition rate was calculated as: Inhibition rate = (Control colony diameter - Treated colony diameter / Control colony diameter) × 100%.

[0024] A strain XS-4, exhibiting good inhibitory effects against the anthracnose causal agent *C. camelliae*, was isolated and screened from tested soil samples using the plate confrontation method. The plate inhibition rate of strain XS-4 against *C. camelliae* was 76.42%. Figure 1 ).

[0025] Example 2 Identification of strain XS-4

[0026] Morphological characteristics

[0027] The single colony morphology of strain XS-4 on Gao's No. 1 medium was round. Both basal and aerial hyphae grew well, with the aerial hyphae being pale pink in color. Microscopic observation showed that the spore chains of strain XS-4 were straight chains, and the spores were round. It could grow on all eight culture media with observed growth characteristics, showing the best growth on ISP2 and Gao's No. 1 medium. No aerial hyphae were observed on ISP4 and nutrient agar medium. Soluble pigments were produced on ISP2, ISP6, and PDA mediums, but no soluble pigments were produced on other media. Figure 2 (Table 1)

[0028] Table 1. Culture characteristics of antagonistic strain XS-4 on different culture media

[0029] Yeast extract oat culture medium ISP2 +++ yellowish brown White light yellow Inorganic salt starch agar ISP4 + White none none ISP6 ISP6 medium ++ grey purple gray pale yellow Gao's No. 1 +++ White light purple none Czapek's medium + pale yellow pinkish white none Bennett medium ++ light yellow White none Nutrient agar medium + grey none none Potato glucose agar medium ++ pale yellow-green pinkish white light purple

[0030] Note: +++ indicates dense colonies with well-developed aerial hyphae; ++ indicates relatively dense colonies with relatively well-developed aerial hyphae; + indicates sparse colonies with poorly developed aerial hyphae.

[0031] Physiological and biochemical characteristics

[0032] Physiological and biochemical tests showed that strain XS-4 was positive for gelatin liquefaction, milk coagulation and peptonization, catalase, and arginine decarboxylase, but negative for methyl red test, VP test, H2S production, and nitrate reductase. It can utilize L-arginine, L-glycine, L-alanine, and L-tryptophan, but cannot utilize L-cysteine ​​and L-glutamic acid. Strain XS-4 can grow at a 4% NaCl concentration and its pH tolerance range is 5-10 (see Table 2).

[0033] Table 2 Physiological and biochemical characteristics of antagonistic strain XS-4

[0034] NaCl tolerance 4％ Gelatin liquefaction + pH tolerance 5-10 Methyl red test - L-arginine + Arginine decarboxylase + L-glycine + catalase test + L-cysteine - Acetylmethylation test (VP test) - L-alanine + nitrate reductase - L-Tryptophan + Milk coagulation + L-glutamic acid - milk peptone + <![CDATA[Hydrogen sulfide-producing H2S]]> -

[0035] Note: +: positive; -: negative

[0036] Molecular biological identification

[0037] The 16S rRNA gene sequence of strain XS-4 was obtained by PCR amplification. Sequencing yielded a 1421 bp gene fragment. This sequence was submitted to GenBank for BLAST alignment analysis. A phylogenetic tree was constructed using gene sequences with high similarity. The results showed that strain XS-4 clustered with *Streptomyces polychromogenes*. Therefore, based on morphological characteristics, physiological and biochemical characteristics, and the 16S rRNA gene sequence phylogenetic tree results, strain XS-4 was identified as *Streptomyces polychromogenes*. Figure 3 .

[0038] The 16S rRNA gene sequence is as follows:

[0039]

[0040] Example 3: Determination of the antibacterial spectrum of Streptomyces multiflora XS-4

[0041] The antibacterial spectrum of strain XS-4 was determined using the plate confrontation method. Eight fungal discs of the tested plant pathogens, each 5 mm in diameter, were placed in the center of a PDA plate using a 5 mm punch. An antagonistic bacterium was then picked up with a sterile inoculation loop and streaked on both sides at a distance of 15 mm from the pathogen. A control was prepared by inoculating only the pathogens. Each treatment was repeated three times. After incubation at 28℃ for 7 days, the diameter of the inhibition zone was measured. The inhibition rate of antagonistic strain XS-4 against the eight tested plant pathogens—*Verticillium dahlia* (cotton wilt causal agent), *Phytophtherafragariae* (strawberry blight causal agent), *Fusarium oxysporum* (pepper wilt causal agent), *Diaporthe citri* (citrus scab causal agent), *Exserohilum turcicum* (corn leaf spot causal agent), *Fusarium oxysporum* (water chestnut causal agent), *Colletotrichum circinans* (Polygonatum anthracnose causal agent), and *Phomopsis vexans* (eggplant brown spot causal agent)—was calculated using the formula in section 1.2.

[0042] The antibacterial spectrum determination results of *Streptomyces polymorpha* XS-4 showed that it had good inhibitory effects on eight other plant pathogens, with inhibition rates all above 50%. The inhibition rates against strawberry blight and eggplant brown spot were both greater than 70%, but the inhibitory effect against strawberry blight was the best, with an inhibition rate of 75.23±0.73. The inhibition rate against cotton wilt pathogen was the lowest, but it still reached 53.79±1.74. Therefore, it is indicated that *Streptomyces polymorpha* XS-4 has broad-spectrum antibacterial activity and good antibacterial effect, as detailed in Table 3.

[0043] Table 3. Determination of the antibacterial spectrum of Streptomyces polymorpha XS-4 against 8 plant pathogens.

[0044] strawberry blight 75.23±0.73a Polygonatum anthrax bacteria 69.31±0.57bc Citrus scab 64.26±1.49d Eggplant brown spot disease 71.10±1.04b Fusarium wilt of peppers 66.04±1.49cd Fusarium oxysporum 64.48±0.66d cotton wilt 53.79±1.74e Maize leaf spot 69.50±0.79bc

[0045] Note: Different lowercase letters after the data in the table indicate significant differences at the P<0.05 level.

[0046] Example 4: Effect of Streptomyces multiflora XS-4 on the mycelial growth of *Streptomyces thuringiensis* (the causal agent of anthracnose).

[0047] The anthracnose pathogen C. camelliae of tea was co-cultured with the antagonistic bacterium XS-4 in a 28℃ incubator for 5 days using the plate confrontation method. The pathogen was allowed to grow naturally for 5 days without the antagonistic bacterium as a control. The hyphae of the anthracnose pathogen of tea were scraped from the edge of the inhibition zone for scanning electron microscopy observation and photography.

[0048] Scanning electron microscopy results showed that co-culturing *Streptomyces multiflora* XS-4 with *C. camelliae* inhibited the mycelial growth of *C. camelliae*. Compared with the control group, the most obvious effect in the treatment group was that the mycelia were tightly intertwined, and the mycelia became deformed, wrinkled, and shriveled. Figure 4 .

[0049] Example 5: Determination of antibacterial activity of Streptomyces multiflora XS-4

[0050] Optimization of fermentation medium for Streptomyces xS-4

[0051] Twenty mycelial cakes, each 5 mm in size, were selected and placed in 200 mL of millet medium, LB, KMB, YEPD, ISP2, PDB, and corn medium, respectively. After incubation at 28℃ and 180 r / min for 7 days, the aseptic fermentation was centrifuged at 12000 r / min. The supernatant was then filtered through a 0.22 μm microporous membrane to obtain the aseptic fermentation broth. The aseptic fermentation broth was mixed with PDA at a ratio of 1:9, poured onto plates, and inoculated with *C. camelliae*. After incubation at 28℃ for 5 days, the colony diameter was measured, and the inhibition rate was calculated according to the formula in section 1.2.

[0052] The results of the optimization experiment of fermentation medium for *Streptomyces polymorpha* XS-4 showed that the sterile fermentation filtrate of strain XS-4 exhibited the highest antibacterial activity against *Anthracis aureus* var. *tea* after fermentation on KMB medium, with an inhibition rate of 67.73±0.72%. The second best medium was ISP2, with an inhibition rate of 66.08±0.61%. In contrast, the sterile fermentation filtrates from corn, millet, and PDB media showed very low inhibition rates against *Anthracis aureus* var. *tea*, with the sterile filtrate from corn medium showing an inhibition rate of only 3.35±0.81%. Therefore, the optimal fermentation medium for strain XS-4 is KMB medium, as the antibacterial substances obtained from fermentation on this medium showed the best antibacterial effect against *Anthracis aureus* var. *tea*. Figure 5 .

[0053] Optimization of fermentation time for strain XS-4

[0054] After fermentation in KMB medium for 4, 5, 6, 7, 8, and 9 days, the aseptic fermentation filtrate of strain XS-4 did not significantly affect the antibacterial activity against *Anthracis aureus*, the pathogen causing tea anthracnose. However, with increasing culture time, the highest antibacterial activity was observed at 7 days, with an inhibition rate of 68.02 ± 0.81% against *Anthracis aureus*. The inhibition rate of the aseptic filtrate decreased slightly at 8 days, but the decrease was not significant, remaining above 60%. Figure 6 .

[0055] Stability of sterile fermentation broth of strain XS-4

[0056] The results of acid-base stability, temperature stability, UV stability, and protease stability tests on the aseptic fermentation broth of strain XS-4 showed that when pH < 5, its antibacterial activity decreased significantly with decreasing pH value; and when pH > 9, its antibacterial activity decreased significantly with increasing pH value. This indicates that both strong alkalis and strong acids affect its antibacterial activity. Figure 7 A); It exhibits good stability under ultraviolet irradiation; its sterile fermentation broth, after 5 hours of ultraviolet irradiation, still maintains an inhibition rate of over 55% against tea anthracnose bacteria. Figure 7 B); and it has good thermal stability. After treatment at 120℃, the aseptic fermentation broth still showed an inhibition rate of more than 50% against tea anthracnose bacteria. Figure 7 C); It is relatively stable against proteinase K and pepsin, with little fluctuation in antibacterial activity, but it is relatively more stable against pepsin. Figure 7 D).

[0057] Determination of antibacterial properties of strain XS-4

[0058] Determination of the ability of strain XS-4 to produce degrading enzymes

[0059] The detection of amylase, protease, β-1,3-glucanase, and cellulase in strain XS-4 was performed using a three-point inoculation method. The strain was inoculated onto amylase medium, protease medium, APB medium, cellulase medium, and colloidal chitin medium, respectively. After incubation at 28°C for several days, the amylase medium plates were stained with Lugol's iodine solution to observe the presence or absence of hydrolysis zones. For protease, β-1,3-glucanase, and chitinase, the presence or absence of hydrolysis zones was directly observed on the plates. For cellulase, Congo red staining was used first, followed by destaining with NaCl solution, and then the presence or absence of hydrolysis zones was observed.

[0060] The extracellular enzyme activity of strain XS-4 was measured on a plate. On starch hydrolysis medium, strain XS-4 produced a very clear hydrolysis zone, indicating its ability to produce starch hydrolytic enzymes. Figure 8 A); On protease medium, a very clear zone is produced, indicating that strain XS-4 has the ability to produce protease. Figure 8 B); On APB medium, strain XS-4 produced a very obvious hydrolysis zone, indicating that strain XS-4 can produce β-1,3-glucanase (B); Figure 8 C); On carboxymethyl cellulose medium, after Congo red staining and NaCl decolorization, a relatively obvious hydrolysis zone was also produced, indicating that strain XS-4 has the ability to produce cellulase. Figure 8 D); however, no hydrolysis zone was produced on the chitin medium, indicating that it could not produce chitinase.

[0061] Example 6: Detection of nonribosomal polypeptide synthase nrps and polymerase pks genes in strain XS-4

[0062] Using genomic DNA from strain XS-4 as a template, primers A3F: 5′-GCSTACSYSATSTACACSTCSGG-3′ (as shown in SEQ ID NO.2); A7R: 5′-SASGTCVCCSGTSCGGTAS-3′ (as shown in SEQ ID NO.3) were used for PCR amplification of the nrps gene, with primers K1: 5′-TSAAGTCSAACATCGGBCA-3′ (as shown in SEQ ID NO.4); M6R: 5′-CGCAGGTTSCSGTACCAGTA-3′ (as shown in SEQ ID NO.5) used for PCR amplification of the pks-Ⅰ gene, with primers F: 5′-TSGCSTGCTTGGAYGCSATC-3′ (as shown in SEQ ID NO.6) and R: 5′-TGGAANCCGCCGAABCCGCT-3′ (as shown in SEQ ID NO.2). NO.7 shows the PCR amplification of the pks-II gene; the reaction parameters are: 95℃ for 3 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 2 min, 30 cycles; 72℃ for 5 min; the PCR amplification products are detected by 1.5% agarose gel electrophoresis.

[0063] PCR amplification of the nrps and pks genes in strain XS-4 revealed the presence of pks-Ⅰ and pks-Ⅱ genes, but the nrps gene was not detected. Figure 9 This indicates that strain XS-4 possesses a polyketide synthase-related gene, thereby producing related antimicrobial substances.

[0064] Example 7: Control effect of strain XS-4 on anthracnose in indoor potted tea plants.

[0065] Two-year-old tea cultivar Chuyeqi was selected as the experimental material. Surfactants were added to the antagonistic bacterial fermentation broth and mixed thoroughly. Fresh, healthy tea leaves without disease spots were selected. The leaves were punctured with a sterile needle, and the antagonistic bacterial strain XS-4 fermentation broth was sprayed onto the leaves until liquid dripped from them. After drying, a 0.5 cm diameter anthracnose fungal cake was inoculated at the wound site, and the wound was kept moist with cotton. A control group was used, with no antagonistic bacterial fermentation broth treatment. Each treatment was repeated three times, with four tea seedlings in each group. The seedlings were cultured under natural conditions, and the disease incidence was observed. Once disease appeared, the fungal cake was removed. On day 7, the treatment groups were sprayed with the antagonistic bacterial broth again. After 14 days, the diameter of the lesions was investigated and measured to classify the disease severity.

[0066] The disease severity grading standards are as follows: Grade 0: Infection at the puncture site, but not confluent; Grade 1: Confluent lesions, diameter less than 0.5 cm; Grade 2: Lesion diameter 0.5–0.7 cm; Grade 3: Lesion diameter 0.7–1 cm; Grade 4: Lesion diameter 1–1.5 cm; Grade 5: Lesion diameter greater than 1.5 cm. Disease index = ∑(Number of disease-grade spots × Disease grade value) / (Number of inoculation points × Highest grade value) × 100. Control efficacy (%) = (Disease index of control group – Disease index of treatment group) / Disease index of control group × 100

[0067] The pot experiment of antagonistic strain XS-4 (Table 4) showed that, 14 days after inoculation with the pathogen, the disease index of the control group was 32.05; the disease index of the undiluted solution treatment group was 16.39, with a control effect of 48.86% against tea anthracnose; the disease index of the fermentation broth diluted 10 times was 19.89, with a control effect of 37.94%; as the dilution ratio increased, the disease index increased and the control effect decreased, with the disease index of the fermentation broth diluted 100 times being 22.53 and the control effect being 29.70%.

[0068] Table 4. Control effect of strain XS-4 on anthracnose in indoor potted plants.

[0069] CK 32.05±0.92a - Original solution 16.39±0.49d 48.86±1.28a 10x fermentation liquid 19.89±0.81c 37.94±1.89b 100x fermentation liquid 22.53±0.96b 29.70±2.34c

[0070] Note: Data are expressed as mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences (P < 0.05).

[0071] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0072] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polychromogenic Streptomyces XS-4, characterized in that, The Streptomyces griseus ( Streptomycespolychromogenes ) XS-4 was preserved in the China Center for Type Culture Collection on October 19, 2023, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20231945.​ 2. The application of the aseptic fermentation broth, bacterial suspension, and bacterial agent of Streptomyces xS-4 as described in claim 1 in the prevention and control of anthracnose pathogens in tea trees.

3. The application of the polychromogenic Streptomyces XS-4 according to claim 1 in the control of other plant pathogens, wherein the other plant pathogens include strawberry blight fungus, Solomon's seal anthracnose fungus, citrus scab fungus, eggplant brown spot fungus, pepper wilt fungus, water chestnut Fusarium, cotton wilt fungus and corn leaf blight fungus.

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